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2 protocols using mscarlet 1

1

Fluorescent Protein Transfection Protocol

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FusionRed-F-tractin has been previously described [3 (link)]. EGFP-F-tractin (no. 58473), mEmerald (no. 53975), LifeAct-mEmerald (no. 54148), mScarlet-I (no. 85044), and LifeAct-mScarlet-I (no. 85054) were obtained from Addgene (Watertown, MA). 1 μg of plasmid was used to transfect 750,000 cells in each well of a 6-well plate using Lipofectamine 2000 (5 μL; Thermo Fisher Scientific) in OptiMEM (400 μL; Thermo Fisher Scientific). After 30 min at room temperature, plasmid in Lipofectamine 2000/OptiMEM was then incubated with cells in complete media (2 mL) overnight.
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2

Cloning and Expression of RAB34 Constructs

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Gibson assembly, Gateway cloning, and standard molecular biology were used for DNA cloning. Human RAB34 cDNA was obtained from GE Healthcare (MHS6278-202807876) and cloned into Gateway pENTR plasmid pDONR221 (Invitrogen); RAB34 mutants were prepared by site-directed mutagenesis of pENTR-Rab34. Plasmids for tetracycline-inducible expression of LAP-Rab34 (LAP consists of GFP, TEV protease site, and S-tag) were constructed by modification of pCW-Cas9 (Addgene #50661, gift from Eric Lander and David Sabatini). Stable expression of fluorescently tagged ciliary and centriolar proteins was achieved by modification of pCW-Cas9 using cDNAs for the PGK promoter, ARL13B (Addgene #40879, gift from Tamara Caspary), EHD1 (gift from Chris Westlake), Rab8 (gift from Maxence Nachury), and Centrin2 (gift from Tim Stearns), miRFP-670 (Addgene #79987, gift from Vladislav Verkhusha), and mScarlet-I (Addgene #85044, gift from Dorus Gadella). Plasmids for CRISPR-based knockout were constructed using pMCB320 (Addgene #89359, gift from Michael Bassik) and pMJ179 (Addgene #89556, gift from Jonathan Weissman); see Key Resources Table for sgRNA sequences. Plasmids for transient transfection of RAB34 were made by Gateway cloning using pEF5-FRT-LAP-DEST55 (link),56 (link); for bacterial protein expression, RAB34 variants were cloned into pGEX-6P1 (GE Healthcare).
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